Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

ABSTRACT

The present invention describes transgenic animals with human(ized) immunoglobulin loci and transgenes encoding human(ized) Igα and/or Igβ sequences. Of particular interest are animals with transgenic heavy and light chain immunoglobulin loci capable of producing a diversified human(ized) antibody repertoire that have their endogenous production of Ig and/or endogenous Igα and/or Igβ sequences suppressed. Simultaneous expression of human(ized) immunoglobulin and human(ized) Igα and/or Igβ results in normal B-cell development, affinity maturation and efficient expression of human(ized) antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/272,051, filed on Sep. 21, 2016, which in turn is a Divisional ofU.S. patent application Ser. No. 12/972,290, filed Dec. 17, 2010, whichis a Continuation of U.S. patent application Ser. No. 14/895,910, filedAug. 27, 2007, which claims priority under 35 USC Section 119(e) and thebenefit of U.S. Provisional Patent Application No. 60/841,890, filedSep. 1, 2006, the entire disclosures of which are hereby incorporated byreference in their entireties.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The instant application contains a. Sequence Listing which has beensubmitted electronically in ASCII format and is herein incorporated byreference in its entirety. Said ASCII copy, created on Oct. 2, 2018, isnamed SequenceListing.txt and is 10.2 KB in size.

FIELD OF THE INVENTION

This invention relates to a method to improve the expression ofhuman(ized) immunoglobulin in non-human transgenic animals by promotingnormal B-cell development and by sustaining the expression ofhuman(ized) antibodies in non-human animals harboring human(ized)immunoglobulin loci. In particular, this invention relates to thesimultaneous expression of transgenes encoding human(ized) Igα and/orIgβ, components of the B-cell receptor, and transgenes encoding ahuman(ized) immunoglobulin locus or loci. This method allows for thedominant expression of human(ized) antibodies, for example in the blood,milk or eggs of the transgenic non-human animals.

DESCRIPTION OF THE RELATED ART

Antibodies are an important class of pharmaceutical products that havebeen successfully used in the treatment of various human diseases andconditions, such as cancer, allergic diseases, prevention of transplantrejection and host-versus-graft disease.

A major problem of the antibody preparations obtained from non-humananimals is the intrinsic immunogenicity of non-human immunoglobulins inhuman patients. In order to reduce the immunogenicity of non-humanantibodies, it has been shown that by fusing animal variable (V) regionexons with human constant (C) region exons, a chimeric antibody gene canbe obtained. Such chimeric or humanized antibodies have minimalimmunogenicity to humans and are appropriate for use in the therapeutictreatment of human subjects.

Humanized monoclonal antibodies have been developed and are in clinicaluse. However, the use of monoclonal antibodies in general, whetherchimeric, humanized or human, for the treatment of devastating diseasessuch as cancer or infections with virulent pathogens, is limited due tothe complexity, multifactorial etiology and adaptivity of thesediseases. Monoclonal antibodies directed against singularly definedtargets usually fail when those targets change, evolve and mutate. Forinstance, malignancies may gain resistance to standard monoclonalantibody therapies. A solution to this problem is to use polyclonalantibodies which have the ability to target a plurality of evolvingtargets. Polyclonal antibodies can neutralize bacterial or viral toxins,and direct immune responses to kill and eliminate pathogens.

Accordingly, there is a great clinical need for suitable methods for thelarge-scale production of high-titer, high-affinity, humanizedpolyclonal and monoclonal antibodies. Further, since production ofantibodies in larger transgenic animals like rabbits, chickens, sheepand cows is favored from the standpoint of antibody yield, creation oflarger founder animals expressing higher amounts of transgene-encodedproducts is also highly desirable.

Humanized monoclonal antibodies are typically human antibodies in whichsome CDR residues, and possibly some FR residues, are substituted byresidues from analogous sites in non-human, animal, e.g. rodent,antibodies. Humanization can be essentially performed following themethod of Winter and co-workers (Jones et al., Nature, 321: 522 (1986);Riechmann et al., Nature, 332: 323 (1988); Verhoeyen et al., Science,239: 1534 (1988)), by substituting non-human, animal CDRs or CDRsequences (e.g. rodent), for the corresponding sequences of a humanmonoclonal antibody.

While making humanized antibodies in animals, one problem encountered isthe endogenous production of host antibody over transgenic antibody,which needs to be suppressed. It has been described that the homozygousdeletion of the antibody, heavy-chain joining region (JH) gene inchimeric and germ-line mutant mice, results in the complete inhibitionof endogenous antibody production. Transfer of a human germ-lineimmunoglobulin gene array into such germ-line mutant mice will result inthe production of human antibodies upon antigen challenge. See, e.g.,Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993);Jakobovits et al., Nature, 362: 255 (1993); Bruggemann et al., Year inImmunol., 7: 33 (1993); U.S. Pat. No. 7,064,244 issued Jun. 20, 2006;the disclosures of which are incorporated herein by reference in theirentirety.

The introduction of human immunoglobulin genes into the genome of miceresults in the expression of a diversified human antibody repertoire inthese genetically engineered mice. The generation of mice expressinghuman-mouse chimeric antibodies has been described by Pluschke et al.,Journal of Immunological Methods 215: 27-37 (1998). The generation ofmice expressing human immunoglobulin polypeptides has been described byNeuberger et al., Nature 338: 350-2 (1989); Lonberg et al., Int. Rev.Immunol. 13(1):65-93 (1995); and Bruggemann et al., Curr. Opin.Biotechnol., 8(4): 455-8 (1997); U.S. Pat. No. 5,545,806, issued August1996; U.S. Pat. No. 5,545,807, issued August 1996 and U.S. Pat. No.5,569,825, issued October 1996; the disclosures of which areincorporated herein by reference in their entirety. The generation ofcows expressing human antibodies has been described by Kuroiwa et al.,Nature Biotech 20(9): 889-894 (2002). The production of non-humantransgenic animals expressing human(ized) immunoglobulin transloci andthe production of antibodies from such transgenic animals have also beendescribed in detail in PCT Publication Nos. WO 92/03918, WO 02/12437,and in U.S. Pat. Nos. 5,814,318, and 5,570,429, the disclosures of whichare hereby expressly incorporated by reference in their entirety. Thehumanized antibodies obtained have minimal immunogenicity to humans andare appropriate for use in the therapeutic treatment of human subjects.

While the genetic engineering approaches cited above result in theexpression of human immunoglobulin polypeptides in geneticallyengineered mice, the level of human immunoglobulin expression is lowerthan normal. This may be due to species-specific regulatory elements inthe immunoglobulin loci that are necessary for efficient expression ofimmunoglobulins. As demonstrated in transfected cell lines, regulatoryelements present in human immunoglobulin genes may not function properlyin non-human animals. Several regulatory elements in immunoglobulingenes have been described. Of particular importance are enhancersdownstream (3′) of heavy chain constant regions and intronic enhancersin light chain genes. In addition, other, yet to be identified, controlelements may be present in immunoglobulin genes. Studies in mice haveshown that the membrane and cytoplasmic tail of the membrane form ofimmunoglobulin molecules play an important role in expression levels ofhuman-mouse chimeric antibodies in the serum of mice homozygous for thehuman Cγ1 gene. Therefore, for the expression of heterologousimmunoglobulin genes in animals, it is desirable to replace sequencesthat contain enhancer elements and exons encoding transmembrane (M1exon) and cytoplasmic tail (M2 exon) with sequences that are normallyfound in the animal in similar positions.

Human immunoglobulin expression in these genetically engineered animalsmay also be affected by B-cell development of the non-human B-cellscarrying the human or humanized immunoglobulin loci. The influence ofthe B-cell receptor (BCR) on B-cell development has been studiedextensively in mice. However, it has been unclear how a human orpartially human antibody combines to form a functional BCR, and whethersuch a BCR would efficiently influence the development and survival ofnon-human B-cells expressing human(ized) Ig, in transgenic animals,which, in turn, would affect antibody yields.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an amino acid alignment of the human Igα polypeptidesequence (SEQ ID NO: 1) with other non-human Igα sequences (SEQ ID NOs:2-6).

FIG. 2 shows an amino acid alignment of the human Igβ polypeptidesequence (SEQ ID NO: 7) with other non-human IGβ sequences (SEQ ID NOs:8-12).

SUMMARY OF THE INVENTION

In one aspect, the invention provides a transgene construct encoding achimeric Igα subunit of the BCR, wherein the chimeric Igα subunitcomprises an intracellular domain sequence and a transmembrane domainsequence of a non-human, Igα polypeptide sequence; and further, apolypeptide having at least 85% sequence identity to the extracellulardomain of human Igα of SEQ ID NO.: 1.

In a second aspect, the invention provides a transgene constructencoding a chimeric Igβ subunit of the BCR, wherein the chimeric Igβsubunit comprises an intracellular domain sequence and a transmembranedomain sequence of a non-human, Igβ polypeptide sequence, and further, apolypeptide having at least 85% sequence identity to the extracellulardomain of the human Igβ of SEQ ID NO.:7.

In a certain embodiment of the invention, the type of non-human Igαpolypeptide sequence includes, but is not limited to, the bovine (SEQ IDNO: 2); murine (SEQ ID NO: 3); canine (SEQ ID NO: 4); primate ((SEQ IDNO: 5); rabbit (SEQ ID NO: 6) or other non-human sequences. In anotherembodiment of the invention, the type of non-human Igβ polypeptidesequence includes, but is not limited to, the canine (SEQ ID NO: 8); rat(SEQ ID NO: 9); bovine (SEQ ID NO: 10); murine (SEQ ID NO: 11); chicken(SEQ ID NO: 12) or other non-human sequences.

In a third aspect, the non-human transgenic animal comprises (a) atransgene construct encoding either a full-length, human Igα subunit ofSEQ ID NO.: 1, or the chimeric Igα subunit as defined above, and/or, (b)a transgene construct encoding either a full-length, human Igβ subunitof SEQ ID NO.: 7, or the chimeric Igβ subunit, as defined above, and,(c) a transgene construct encoding a human(ized) immunoglobulin locus,wherein the resultant transgene products combine to form a human(ized)B-cell receptor complex.

In one embodiment of this aspect, the expression of any endogenous Igproduction, and/or, endogenous Igα and/or endogenous Igβ subunitexpression, of the non-human transgenic animal is substantially reduced.

In another embodiment, the non-human transgenic animal is selected froma group consisting of rabbit, mouse, rat, pig, sheep, goat, bird, horse,donkey and cow. In a preferred embodiment, the non-human transgenicanimal is a rabbit.

In a fourth aspect, the invention also provides an isolated human(ized)immunoglobulin from the non-human transgenic animal defined above, whichis either an antibody or an antibody fragment. In a certain embodimentof this aspect, the isolated human(ized) immunoglobulin is either apolyclonal or a monoclonal antibody, or alternately, is an antibodyfragment. The antibody fragment can be either from a polyclonal or amonoclonal antibody. Further, the antibody or the antibody fragment canbe labeled, or fused to a toxin to form an immunotoxin, or coupled to atherapeutic agent, or fused to any heterologous amino acid sequencewell-defined and used in the art. In some embodiments, the antibodyfragment is a Fc, Fv, Fab, Fab′ or F(ab′)₂ fragment.

In a fifth aspect, the invention provides an isolated B-cell from thenon-human transgenic animal defined above, where the B-cell expresseseither the native human Igα subunit or a chimeric Igα subunit and/oreither the native human Igβ subunit or the chimeric Igβ subunit, andfurther, also expresses the human(ized) immunoglobulin locus. In certainembodiments, this B-cell is immortalized and in a preferred embodiment,is derived from a rabbit.

In a sixth aspect, the invention provides an antibody preparationcomprising an antibody or an antibody fragment, as described above.

In a seventh aspect, the invention provides a pharmaceutical compositioncomprising an antibody or antibody fragment, as described above, in amixture with a pharmaceutically acceptable ingredient. Thepharmaceutical composition can comprise either a monoclonal antibody ora fragment thereof, or, one or a plurality of polyclonal antibodies orfragments thereof.

In an eighth aspect, the invention provides a method for producinghuman(ized) antibodies in a non-human animal comprising: (a) introducingand expressing a transgene construct encoding either a native human Igαsubunit or a chimeric Igα subunit, and/or a transgene construct encodingeither a native human Igβ subunit or a chimeric Igβ subunit into thenon-human animal; and, (b) introducing and expressing a transgeneconstruct encoding a human(ized) immunoglobulin locus into the non-humananimal; (c) subjecting the animal to an antigenic stimulus; and (d)isolating human(ized) antibodies from the animal. In a certainembodiment of this aspect, the antibody is either a polyclonal or amonoclonal antibody, or is a fragment of a polyclonal or a monoclonalantibody. Further, the antibody or antibody fragment can either belabeled, or can be fused to a toxin to form an immunotoxin, or coupledto a therapeutic agent, or can be fused to any heterologous amino acidsequence.

In a ninth aspect, the invention provides a method for producing anon-human animal expressing human(ized) antibodies comprising: (a)introducing and expressing a transgene construct encoding either anative human Igα subunit or a chimeric Igα subunit and/or a transgeneconstruct encoding either a native human Igβ subunit or a chimeric Igβsubunit into the B-cell of the non-human animal; and, (b) introducingand expressing a transgene construct encoding a human(ized)immunoglobulin locus into the non-human animal; wherein the resultanttransgene products combine to form a human(ized) B-cell receptorcomplex. In one embodiment, the non-human animal expressing human(ized)antibodies is an animal that creates antibody diversity by geneconversion and/or somatic hypermutation. In a preferred embodiment, theanimal is a rabbit.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanismsand Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provideone skilled in the art with a general guide to many of the terms used inthe present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

“A transgene construct or expression construct” as defined herein,refers to a DNA molecule which contains the coding sequence for at leastone transgene of interest along with appropriate regulatory sequencesrequired for temporal, cell specific and/or enhanced expression of thetransgene(s) of interest within target cells of a non-human transgenicanimal.

“B-cells” are defined as B-lineage cells that are capable of undergoingrearrangement of immunoglobulin gene segments and expressingimmunoglobulin genes at some stage in their life cycle. These cellsinclude, but are not limited to, early pro-B-cells, late pro-B-cells,large pre-B-cells, small pre-B-cells, immature B-cells, mature B-cells,memory B-cells, plasma cells, etc.

“B-cell receptor (BCR) complex” as defined herein, refers to themultisubunit immune recognition receptor expressed on B-cells, whichincludes the following subunits: the antigen (Ag) receptor, themembrane-bound immunoglobulin (mIg), the Igα subunit and the Igβsubunit. The B-cell receptor, its components, and its association withthe five immunoglobulin classes have been described by Wienands et al.,EMBO J. 9(2): 449-455 (1990), Venkitaraman et al., Nature 352: 777-781(1991), Herren et al., Immunologic Res. 26(1-3): 35-43 (2002). Inaddition, there are several BCR-associated proteins (BAPs) that havebeen cloned and sequenced, but their function(s) remain unknown, andtheir role, as components of the BCR, has been questioned.BCR-associated proteins have been described by Adachi et al., EMBO J15(7): 1534-1541 (1996) and Schamel et al., PNAS 100(17): 9861-9866(2003).

“Native Igα or Igβ subunits” refer to naturally occurring Igα or βpolypeptide sequences, which include naturally occurring alleles of Igαor Igβ subunits found in a given type of animal, or in a relatedspecies. These are also referred to as “full-length Igα or Igβsequences”. The human Igα polypeptide sequence was cloned by Flaswinkelet al., Immunogenetics 36 (4): 266-69 (1992); Accession number M74721(FIG. 1, SEQ ID NO: 1). The human Igβ polypeptide sequence was cloned byMueller et al., Eur. J. Biochem. 22, 1621-25 (1992); Accession numberM80461 (FIG. 2, SEQ ID NO: 7).

The term “human(ized)” refers to an entirely human sequence or asequence containing one or more human sequences. Thus, the term, as usedherein, includes human and humanized sequences.

A “chimeric Igα” subunit or protein or polypeptide refers to an Igαpolypeptide sequence from an animal (e.g.; rat, mouse, human, rabbit,chicken, etc.), in which one or more domains of the Igα polypeptide arereplaced with a corresponding domain or domains from a different Igαpolypeptide of another animal or species, or with a corresponding domainor domains from a different allelic Igα version, or from a variant Igαsequence with one or more amino acid substitutions, or from a variantIgα sequence having at least 85% sequence identity to the correspondingdomain of a given Igα sequence. The terms “chimeric Igα” and“human(ized) Igα” are used interchangeably throughout the specification.Igα polypeptide sequences (SEQ ID NOs: 2-6) from some non-human animalsare also defined in FIG. 1.

A “chimeric Igβ” subunit or protein or polypeptide refers to an Igβpolypeptide sequence from an animal (e.g.; rat, mouse, human, rabbit,chicken, etc.), in which one or more domains of the Igβ polypeptide arereplaced with a corresponding domain or domains from a different Igβpolypeptide of another animal or species, or with a corresponding domainor domains from a different allelic Igβ version, or from a variant Igβsequence with one or more amino acid substitutions, or from a variantIgβ sequence having at least 85% sequence identity to the correspondingdomain of a given Igβ sequence. The terms “chimeric Igβ” and“human(ized) Igβ” are used interchangeably throughout the specification.Igβ polypeptide sequences (SEQ ID NOs: 8-12) from some non-human animalsare defined in FIG. 2.

“Intracellular polypeptide or domain” or “cytoplasmic tail” refers tothat part of the polypeptide sequence of a given membrane-bound proteinor subunit that exists within the bounds of the cell. Usually, theintracellular domain of the protein is responsible for signaltransduction.

By “intracellular domain sequence” of an Igα or Igβ subunit is meant thepolypeptide sequence of the Igα or Igβ polypeptide, or fragmentsthereof, that usually exist within the bounds of the cell.

“Transmembrane domain sequence” of an Igα or Igβ subunit is meant thepolypeptide sequence of the Igα or Igβ polypeptide, or fragments thereofthat spans a biological membrane such as a plasma membrane, organellemembrane, or lipid bilayer. The “transmembrane domain sequence” asdefined herein includes naturally occurring membrane-spanningpolypeptides, or can be non-naturally occurring consensus sequences, orfragments thereof.

“Extracellular polypeptide or domain” refers to that part of thepolypeptide sequence of a given membrane-bound protein or subunit thatusually exists outside the bounds of the cell. By “extracellular domainof Igα or Igβ” is meant the polypeptide sequence of the Igα or Igβpolypeptide, or fragments thereof, that exist outside the bounds of thecell.

The term “human(ized) immunoglobulin locus” as used herein includes bothnaturally occurring sequences of a human immunoglobulin or Ig gene locusor a segment thereof, degenerate forms of naturally occurring sequencesof a human Ig gene locus or segments thereof, as well as syntheticsequences that encode a polypeptide sequence substantially identical toa polypeptide encoded by a naturally occurring sequence of a human Iggene locus or a segment thereof. In a particular embodiment, the humanIg gene segment renders the immunoglobulin molecule non-immunogenic inhumans. Here, the terms “human(ized) or humanized immunoglobulin (Ig)heavy and/or light chain locus” or “human or human(ized) immunoglobulinor Ig locus” are used interchangeably.

The term “human(ized) B-cell receptor (BCR) complex” as used hereinrefers to those multisubunit BCR complexes in which the Igα subunit iseither a native, human Igα subunit or a chimeric Igα subunit havinghuman or humanized Igα sequences as described above; and/or further, inwhich the Igβ subunit is either a native, human Igβ subunit or achimeric Igβ subunit having human or humanized Igβ sequences asdescribed above; and further, where the membrane-bound immunoglobulin(mIg) is that of a human(ized) immunoglobulin, as described above.

The terms “human antibody” and “human immunoglobulin” are used herein torefer to antibodies and immunoglobulin molecules comprising fully humansequences.

The terms “humanized antibody” and “humanized immunoglobulin,” as usedherein, mean an immunoglobulin molecule comprising at least a portion ofa human immunoglobulin polypeptide sequence (or a polypeptide sequenceencoded by a human immunoglobulin gene segment). The humanizedimmunoglobulin molecules of the present invention can be isolated from atransgenic non-human animal engineered to produce humanizedimmunoglobulin molecules. Such humanized immunoglobulin molecules areless immunogenic to primates, especially humans, relative tonon-humanized immunoglobulin molecules prepared from the animal orprepared from cells derived from the animal. Humanized immunoglobulinsor antibodies include immunoglobulins (Igs) and antibodies that arefurther diversified through gene conversion and somatic hypermutationsin gene converting animals. Such humanized Ig or antibodies are not“human” since they are not naturally made by humans (since humans do notdiversify their antibody repertoire through gene conversion) and yet,the humanized Ig or antibodies are not immunogenic to humans since theyhave human Ig sequences in their structure.

By the term “substantially reduced” endogenous Ig production, and/or Igαand/or Igβ subunits expression is meant that the degree of production ofeither the endogenous Ig alone or additionally, endogeous Igα and/or Igβexpression is reduced preferably at least about 30%-49%, or morepreferably at least about 50%-79%, or even more preferably at leastabout 80-89%, or most preferably by about 90-100% in the transgenicanimal.

The term “monoclonal antibody” is used to refer to an antibody moleculesynthesized by a single clone of B-cells.

The term “polyclonal antibody” is used to refer to a population ofantibody molecules synthesized by a population of B-cells.

An “immunoglobulin (Ig) locus” having the capacity to undergo generearrangement and gene conversion is also referred to herein as a“functional” Ig locus, and the antibodies with a diversity generated bya functional Ig locus are also referred to herein as “functional”antibodies or a “functional” repertoire of antibodies.

The term “non-human (transgenic) animal” as used herein includes, but isnot limited to, mammals such as, for example, non-human primates,rodents (e.g. mice and rats), non-rodent mammals, such as, for example,rabbits, pigs, sheep, goats, cows, pigs, horses and donkeys, and birds(e.g., chickens, turkeys, ducks, geese and the like). The term“non-primate animal” as used herein includes, but is not limited to,mammals other than primates, including but not limited to the mammalsspecifically listed above.

DETAILED DESCRIPTION

This invention is based, at least in part, on the recognition that theproduction of human or humanized immunoglobulin (includingimmunoglobulin chains) in a non-human transgenic animal can besignificantly increased by co-expressing human or humanized Igα and/orIgβ in the B cells of the animal. The inclusion of human or humanizedIgα and/or Igβ in the B cells in transgenic animals is believed toreconstitute and improve interactions between the B-cell receptorproteins, thereby enhancing antigen recognition, B-cell development andsurvival of the B cells carrying such transgenes. The co-expression ofhumanized immunoglobulin in transgenic animals already carrying thehuman or humanized Igα and/or Igβ transgenes would vastly improvehumanized immunoglobulin production. It would be additionally desirableto express both, the human or humanized Igα and/or Igβ transgene and thehumanized immunoglobulin transgene against a knockout background of,preferably, both endogenous Ig, as well as endogenous Igα and/or Igβ.

The B-Cell Receptor and Its Associated Proteins

The B-cell receptor consists of membrane bound immunoglobulin and asignal-transducing heterodimer, consisting of two disulfide-linkedglyoproteins called Igα and Igβ. In addition, BCR associated proteins(BAPs) have been described.

Expression of the BCR is important for B-cell development, selection andsurvival. These processes depend on BCR signaling through the Igα/Igβheterodimer. The cytoplasmic domains of these molecules carry a sequencemotif that contains several tyrosine residues assembled into theso-called immunoreceptor tyrosine-based activation motif (ITAM), whichare phosphorylated upon BCR triggering.

Gene targeting experiments have shown that the cytoplasmic domains ofthe Igα/Igβ heterodimer are crucial for B-cell development. Signalstransduced by the Igα/Igβ heterodimer are involved in both positive andnegative selection of developing B-cells.

A membrane bound immunoglobulin (mIg) molecule consists of two heavychains, forming a homodimer, and two light chains, each of which iscovalently bound to one of the heavy chains. At the N-terminus the heavychain carries a VH domain, which, depending on the isotype, is followedby either 4 (IgM, IgE), 3 (IgG, IgA) or 2 (IgD) C-domains. Theantigen-binding site is formed by the hypervariable regions of a VH:VLpair. Thus, each mIg molecule has two antigen binding sites.

The mIgM molecule differs from the secreted form of IgM in that thesecreted IgM forms a pentamer with 10 potential antigen binding sites.The pentamerization is controlled by sequences in the C-terminal part ofthe secreted μs chain. This part, consisting of 22 amino acids, isabsent in the membrane-bound μm chain, which instead carries 48C-terminal amino acids encoded by the M1 and M2 exons.

The μm-specific part of the sequence is the most evolutionarilyconserved part of the whole IgM molecule. It is nearly identical betweenmouse, rabbit and human mIgM, and the conservation is still obvious ifone compares mouse with shark mIgM. Conservation of amino acids is alsoapparent when one compares the C-terminal sequence of mIgM to that ofother mIg isotypes of the mouse. This finding provides evidence that theconserved transmembrane amino acids are interacting either with eachother in the H chain homodimer or with the Igα and Igβ subunits.

Both, Igα and Igβ have a 22 amino acid transmembrane segment, followedby a C-terminal cytoplasmic tail of about 40-70 amino acids, whichcontain several tyrosine residues. At the N-terminus, both proteinscarry a leader peptide, followed by a extracellular domain containingcysteine residues, a tryptophan, as well as several other conservedamino acids found in proteins of the Ig superfamily. This suggests thatthe extracellular parts of both Igα and Igβ subunits form an Ig-likedomain. Besides cysteines that form intra-domain disulfide bonds, theIgα and Igβ sequences contain additional cysteines that presumably forminter-chain disulfide bonds between the Igα and Igβ subunits.

A comparison between the mouse and the human Igα sequence shows that,all residues important for the information of the Ig domain andinter-chain bonds are conserved between the Igα of the two species. Thecomparison, however, also shows that sequence conservation in theextracellular part only amounts to about 56%, while the transmembraneand cytoplasmic tail show conservation of 100% and 87%, respectively.The latter reflects the importance of the residues within the C-terminalpart of the molecule.

The assembly of the mIgM molecule with the Igα/Igβ heterodimer isnecessary for surface expression of mIgM. This requirement can beabolished by mutations of the transmembrane part of the μm chain. Forexample, replacement of the transmembrane region of the μm chain withthe transmembrane part of the H-2K^(κ) molecule results in the surfaceexpression of mIgM independent of Igα/Igβ. These data demonstrate thatthe μm transmembrane region is required for specific interactionsbetween the μm chain and the Igα/Igβ heterodimer. In addition, B-cellshave a control mechanism that prevents transport of single orincompletely assembled components of transmembrane protein complex outof the ER.

Although the transmembrane portions of the BCR are probably the mostimportant structures that are required for the formation of the BCRcomplex, the extracellular Ig-domain of Igα and Igβ has also beensuggested to play a role in the binding of the mIgM molecule. Forinstance, in the mouse cell line J558L μm, which does not express mouseIgα, transfection with an Igα transgene restored the surface expressionof mIgM. Interestingly, transfection with a mouse Igα gene resulted in10-times higher expression than transfection with a human Igα gene. Thisdata suggests that the extracellular domain of Igα, additionally, mayinteract with the extracellular parts of mIgM. On the other hand,transgenic mice with human immunoglobulin loci do express humanimmunoglobulins. It remains unclear whether B-cell development, B-cellsurvival or expression of human(ized) mIgM in transgenic non-humananimals would be influenced by the co-expression of human Igα and/orhuman Igβ in B-cells carrying human(ized) mIgM genes.

In addition, there are several BCR-associated proteins (BAPs) that havebeen cloned and sequenced, but their function(s) remain unknown. Eventhough these proteins are associated with the BCR, their role, ascomponents of the BCR, has been questioned. Yet, the ubiquitousexpression and strong evolutionary conservation of BAPs suggest thatthey must play an important role, possibly in general cellular processesand several putative functions have been proposed. For example, theseproteins may be involved in coupling the BCR to the cytoskeleton, or incontrolling vesicular transport. Lastly, it has been proposed that theyfunction as chaperones, helping in the folding and assembly oftransmembrane proteins.

Relevant Literature

The B-cell receptor, its components, and its association with the fiveimmunoglobulin classes have been described by Wienands et al., EMBO J.9(2): 449-455 (1990), Venkitaraman et al., Nature 352: 777-781 (1991),Herren et al., Immunologic Res. 26(1-3): 35-43 (2002). BCR associatedproteins have been described by Adachi et al., EMBO J 15(7): 1534-1541(1996) and Schamel et al., PNAS 100(17): 9861-9866 (2003). The influenceof the B-cell receptor on B-cell development and survival has beendescribed by Reth, Annual Reviews of Immunology 10: 97-121 (1992), Krauset al., Cell 117(6): 787-800 (2004), Sayegh et al., ImmunologicalReviews 175: 187-200 (2000), Reichlin et al, Journal of ExperimentalMedicine 193(1): 13-23 (2001), Pike et al., Journal of Immunology 172:2210-2218 (2004), Pelanda et al., Journal of Immunology 169: 865-872(2002). Regulation of BCR signaling and its influence in B-celldevelopment and apoptosis have been described in Cronin et al., J.Immunology 161: 252-259 (1998), Muller et al., PNAS 97 (15): 8451-8454(2000), Cragg et al., Blood 100: 3068-3076 (2002), Wang et al., J.Immunology 171: 6381-6388 (2003), Fuentes-Pananá et al., J. Immunology174: 1245-1252 (2005). The disclosures of the above cited references areincorporated herein by reference in their entirety.

The present invention therefore is directed to methods for co-expressinghuman(ized) Igα and/or human(ized) Igβ in B-cells, particularly intransgenic animals that are capable of producing a diversifiedhuman(ized) antibody repertoire to improve B-cell survival in suchtransgenic animals. Types of animals include larger non-human animalslike rabbits, birds, chickens, sheep, goats, cows, swine, horses anddonkeys. When these animals express an Ig translocus, because of theirlarger size, their antibody yields should also be greater. Thus, thisinvention aims at creating larger founder animals producing higheramounts human(ized) immunoglobulins through enhanced B-cell developmentand survival.

Accordingly, the present invention is directed to transgene constructsencoding full-length human Igα and Igβ polypeptides, or, chimerictransgene constructs encoding for chimeric or humanized Igα and chimericor humanized Igβ polypeptides, as defined further below.

By “transgene or transgene construct encoding the human Igα and/or Igβpolypeptide” is meant the native, full length, human Igα and/or Igβ DNAsequence respectively, as well as any variant, codon optimized DNAsequence which encodes for a functionally equivalent polypeptide of Igαor Igβ, but which has a different DNA sequence based on codondegeneracy. This concept is discussed in detail further below. Thenative, full length, human Igα polypeptide sequence is defined in SEQ IDNO: 1 (FIG. 1). The native, full length, human Igβ polypeptide sequenceis defined in SEQ ID NO: 7 (FIG. 1).

Also referred to herein is “nucleic acid molecule or transgene ortransgene construct encoding the chimeric or human(ized) Igα”. A“chimeric Igα” subunit or protein or polypeptide refers to an Igαpolypeptide sequence from an animal (e.g.; rat, mouse, human, rabbit,chicken, etc.), in which one or more domains of the Igα polypeptide arereplaced with a corresponding domain or domains from a different Igαpolypeptide of another animal or species, or with a corresponding domainor domains from a different allelic Igα version, or from a variant Igαsequence with one or more amino acid substitutions, or from a variantIgα sequence having at least 85% sequence identity to the correspondingdomain of a given Igα sequence. The terms “chimeric Igα” and“human(ized) Igα” are used interchangeably throughout the specification.The non-human Igα polypeptide sequences from which the intracellularand/or the transmembrane domain sequences can be obtained, for example,include, but are not limited to, bovine (SEQ ID NO: 2); murine (SEQ IDNO: 3); canine (SEQ ID NO: 4); primate ((SEQ ID NO: 5); rabbit (SEQ IDNO: 6) or other non-human sequences.

Also referred to herein is “nucleic acid molecule or transgene ortransgene construct encoding the chimeric or human(ized) Igβ”. A“chimeric Igβ” subunit or protein or polypeptide refers to an Igβpolypeptide sequence from an animal (e.g.; rat, mouse, human, rabbit,chicken, etc.), in which one or more domains of the Igβ polypeptide arereplaced with a corresponding domain or domains from a different Igβpolypeptide of another animal or species, or with a corresponding domainor domains from a different allelic Igβ version, or from a variant Igβsequence with one or more amino acid substitutions, or from a variantIgβ sequence having at least 85% sequence identity to the correspondingdomain of a given Igβ sequence. The terms “chimeric Igβ” and“human(ized) Igβ” are used interchangeably throughout the specification.The non-human Igβ polypeptide sequences from which the intracellularand/or the transmembrane domain sequences can be obtained, for example,include, but are not limited to, canine (SEQ ID NO: 8); rat (SEQ ID NO:9); bovine (SEQ ID NO: 10); murine (SEQ ID NO: 11); chicken (SEQ. ID NO:12); or other non-human sequences.

Thus, briefly, a chimeric Igα or Igβ transgene consists of 1) anucleotide sequence encoding the extracellular domain of the human Igαor Igβ respectively, and 2) a nucleotide sequence encoding thetransmembrane and the intracellular domain of the Igα or Igβ from thehost transgenic animal, respectively.

In a further aspect, the present invention is also directed totransgenic constructs encoding for a human(ized) immunoglobulins orlocii as described in a previously filed U.S. applications, nowavailable as U.S. Publication No. 2003-0017534, published Jan. 23, 2003and U.S. Publication No. 2006-0026696, published Feb. 2, 2006, thedisclosures of which is hereby incorporated by reference in theirentirety. The transgenic animals, B-cells or cell lines generatedthereof, and the relevant methodologies disclosed therein also form anaspect of this invention.

In an alternative approach to the above mentioned aspect, the presentinvention is also directed to transgenic constructs encoding forhuman(ized) immunoglobulin or Ig chain or loci, as described in U.S.Pat. No. 5,545,806, issued August 1996; U.S. Pat. No. 5,545,807, issuedAugust 1996 and U.S. Pat. No. 5,569,825, issued October 1996, U.S. Pat.No. 7,064,244, issued Jun. 20, 2006; or in PCT Publication Nos. WO92/03918, WO 02/12437, and in U.S. Pat. Nos. 5,814,318, 5,570,429; alsosee Jakobovits et al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993);Jakobovits et al., Nature, 362: 255 (1993); Bruggemann et al., Year inImmunol., 7: 33 (1993); Pluschke et al., Journal of ImmunologicalMethods 215: 27-37 (1998); Neuberger et al., Nature 338: 350-2 (1989);Lonberg et al., Int. Rev. Immunol. 13(1):65-93 (1995); and Bruggemann etal., Curr. Opin. Biotechnol., 8(4): 455-8 (1997); and Kuroiwa et al.,Nature Biotech 20(9): 889-894 (2002) the disclosures of which is herebyincorporated by reference in their entirety. The transgenic animals,B-cells or cell lines generated thereof, and the relevant methodologiesdisclosed therein also form an aspect of this invention.

The transgenes or transgene constructs may be introduced into theanimal's genome by a variety of techniques including microinjection ofpronuclei, transfection, nuclear transfer cloning, sperm-mediated genetransfer, testis-mediated gene transfer, and the like.

In one embodiment, the human Igα and/or Igβ gene, is preferablyexpressed in the B-cells of the transgenic animal by means of animmune-specific promoter, preferably a B-cell specific promoter. Thishuman Igα or Igβ gene expression happens preferably within B-cellsalone, leading to enhanced B-cell development and survival of thenon-human transgenic animal. By “B-cell specific promoter” is meant thepromoter/enhancers sequence of any B-cell specific genes, and/orvariants or engineered portions thereof, that normally controls theexpression of genes expressed in a B-cell, examples of which include,but are not limited to, promoters/enhancers of CD19, CD20, CD21, CD22,CD23, CD24, CD40, CD72, Blimp-1, CD79b (also known as B29 or Ig beta),mb-1 (also known as Ig alpha), tyrosine kinase blk, VpreB,immunoglobulin heavy chain, immunoglobulin kappa light chain,immunoglobulin lambda-light chain, immunoglobulin J-chain, etc. In apreferred embodiment, the CD79a, CD79b, or kappa light chainpromoter/enhancer drives the B-cell specific expression of the human Igαand/or Igβ genes.

In yet another embodiment, the transgene construct comprising thenucleic acid molecule encoding the human Igα and/or Igβ genes iscoexpressed with the transgene construct comprising an exogenousimmunoglobulin or immunoglobulin (Ig) chain transgene locus. In thisembodiment, both the Ig transgene locus and the human Igα and/or Igβtransgene may be present on the same transgenic expression vector or ontwo different transgenic expression vectors. In the latter case, the twotransgenic expression vectors may be introduced into the non-humantransgenic animal either at the same time or sequentially.

In accordance with this invention, variants of the human full length orextracellular domain alone, of Igα or Igβ are included herein. By thisis meant nucleic acid sequences that allow for the degeneracy of thegenetic code, nucleic acid sequences that encode for a polypeptidesequence that comprises amino acid substitutions of functionallyequivalent residues and/or mutations that enhance the functionality ofthe extracellular domain. “Functionality of the extracellular domain”includes, but is not limited to, formation of a BCR capable of signaltransduction

By allowing for the degeneracy of the genetic code, the inventionencompasses sequences that have at least about 70%, more usually about80 to 85%, preferably at least about 90% and most preferably about 95%sequence identity to the extracellular polypeptide sequence of human Igαand human Igβ.

The term biologically functional equivalent is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%; or more preferably, between about81% and about 90%; or even more preferably, between about 91% and about99% identical at the amino acid level are considered functionallyequivalent to human Igα and Igβ, provided the biological activity of theproteins is maintained.

The term functionally equivalent codon is used herein to refer to codonsthat encode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andin its underlying DNA coding sequence, and nevertheless produce aprotein with like properties. It is thus contemplated by the inventorsthat various changes may be made in the DNA sequences of genes withoutappreciable loss of their biological utility or activity, as discussedbelow.

In making such changes, the hydropathic index of amino acids may also beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2) glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still produce a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within.+−0.2 is preferred, those that are within .+−0.1 are particularlypreferred, and those within .+−.0.5 are even more particularlypreferred.

As outlined herein, amino acid substitutions generally are based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take into consideration the variousforegoing characteristics are well known to those of skill in the artand include: arginine and lysine; glutamate and aspartate; serine andthreonine; glutamine and asparagine; and valine, leucine and isoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure (Johnson 1993). The underlying rationale behind the use ofpeptide mimetics is that the peptide backbone of proteins exists chieflyto orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outlined above, to engineer second generation moleculeshaving many of the natural properties of human Igα or Igβ with alteredand improved characteristics.

Thus, variant nucleic acid sequences that encode for human Igα or Igβand functionally equivalent polypeptides of human Igα or Igβ are usefulin this invention.

The transgenic vectors containing the genes of interest may beintroduced into the recipient cell or cells and then integrated into thegenome of the recipient cell or cells by random integration or bytargeted integration.

For random integration, a transgenic vector containing a human Igα orIgβ can be introduced into an animal recipient cell by standardtransgenic technology. For example, a transgenic vector can be directlyinjected into the pronucleus of a fertilized oocyte. A transgenic vectorcan also be introduced by co-incubation of sperm with the transgenicvector before fertilization of the oocyte. Transgenic animals can bedeveloped from fertilized oocytes. Another way to introduce a transgenicvector is by transfecting embryonic stem cells and subsequentlyinjecting the genetically modified embryonic stem cells into developingembryos. Alternatively, a transgenic vector (naked or in combinationwith facilitating reagents) can be directly injected into a developingembryo. Ultimately, chimeric transgenic animals are produced from theembryos which contain the human(ized) Ig transgene integrated in thegenome of at least some somatic cells of the transgenic animal.

In a particular embodiment, a transgene containing a human Igα or Igβ israndomly integrated into the genome of recipient cells (such asfertilized oocyte or developing embryos) derived from animal strainswith an impaired expression of endogenous Igα or Igβ. The use of suchanimal strains permits preferential expression of immunoglobulinmolecules from the human(ized) transgenic Ig locus. Alternatively,transgenic animals with human(ized) Igα and/or Igβ transgenes can bemated with animal strains with impaired expression of endogenous Igαand/or Igβ. Offspring homozygous for impaired Igα and/or Igβ andhuman(ized) Igα and/or Igβ can be obtained. Alternatively, expression ofendogenous Igα and/or Igβ may be inhibited or lowered using antisensetechnology, intracellular anti-Igα and/or Igβ expression, and the like.In one embodiment, the method of choice for the knocking down theendogenous production of Igα and/or Igβ of the host animal is the RNAinterference (RNA_(i)) method, which introduces either double-strandedRNA (ds RNA) or more preferably, short or small interfering RNA duplexes(siRNA) into the B-cells having intracellular host animal Igα and/or Igβnucleic acid sequences. This can be achieved using commerciallyavailable kits, including but not limited to, Block iT™ or Stealth™ RNAkits from Invitrogen Corp.

For targeted integration, a transgenic vector can be introduced intoappropriate animal recipient cells such as embryonic stem cells oralready differentiated somatic cells. Afterwards, cells in which thetransgene has integrated into the animal genome and has replaced thecorresponding endogenous gene by homologous recombination can beselected by standard methods (See for example, Kuroiwa et al, NatureGenetics 2004, June 6). The selected cells may then be fused withenucleated nuclear transfer unit cells, e.g. oocytes or embryonic stemcells, which are totipotent and capable of forming a functional neonate.Fusion is performed in accordance with conventional techniques which arewell established. Enucleation of oocytes and nuclear transfer can alsobe performed by microsurgery using injection pipettes. (See, forexample, Wakayama et al., Nature (1998) 394:369.) The resulting eggcells are then cultivated in an appropriate medium, and transferred intosynchronized recipients for generating transgenic animals.Alternatively, the selected genetically modified cells can be injectedinto developing embryos which are subsequently developed into chimericanimals.

Further, according to the present invention, a transgenic animal capableof producing human(ized) Igα and/or Igβ can also be made by introducinginto a recipient cell or cells, one or more of the recombination vectorsdescribed herein above, one of which carries a human Igα and/or Igβ genesegment, linked to 5′ and 3′ flanking sequences that are homologous tothe flanking sequences of the endogenous Igα and/or Igβ gene segment,then selecting cells in which the endogenous Igα and/or Igβ gene segmentis replaced by the human Igα and/or Igβ gene segment by homologousrecombination, and deriving an animal from the selected geneticallymodified recipient cell or cells.

Similar to the target insertion of a transgenic vector, cellsappropriate for use as recipient cells in this approach includeembryonic stem cells or already differentiated somatic cells. Arecombination vector carrying a human Igα and/or Igβ gene segment can beintroduced into such recipient cells by any feasible means, e.g.,transfection. Afterwards, cells in which the human Igα and/or Igβ genesegment has replaced the corresponding endogenous Igα and/or Igβ genesegment by homologous recombination, can be selected by standardmethods. These genetically modified cells can serve as nuclei donorcells in a nuclear transfer procedure for cloning a transgenic animal.Alternatively, the selected genetically modified embryonic stem cellscan be injected into developing embryos which can be subsequentlydeveloped into chimeric animals.

In a specific embodiment, the transgene constructs of the invention maybe introduced into the transgenic animals during embryonic life bydirectly injecting the transgenes into the embryo or indirectly byinjecting them into the pregnant mother or into the egg-laying hen.Transgenic animals produced by any of the foregoing methods form anotherembodiment of the present invention. The transgenic animals have atleast one, i.e., one or more, human(ized) Igα and/or Igβ gene in thegenome, from which a functional human(ized) Igα and/or Igβ protein canbe produced.

Further, the transgene constructs of the invention, namely, the human orhumanized Igα and/or Igβ transgene and the humanized immunoglobulintransgene, are preferably expressed against a knockout background ofeither one, or more preferably both, the endogenous Ig, as well as theendogenous Igα and/or Igβ knockouts. Thus the transgenic animals of thepresent invention are capable of rearranging the human(ized) Ig loci andefficiently expressing a functional repertoire of human(ized) antibodiesagainst a background that has substantially reduced endogenous Igexpression and more preferably, substantially reduced endogeous Igαand/or Igβ as well. In this context, by “substantially” is meant thedegree of endogenous production, of either endogenous Ig expressionalone or additionally, endogeous Igα and/or Igβ expression is reducedpreferably at least about 30%-49%, or more preferably at least about50%-79%, or even more preferably at least about 80-89%, or mostpreferably by about 90-100%.

The present invention provides transgenic rabbits expressing one or morehuman(ized) Ig loci and human(ized) Igα and/or Igβ, that are capable ofrearranging and gene converting the human(ized) Ig loci, and expressinga functional repertoire of human(ized) antibodies. Preferably, theserabbits, additionally, do not produce substantial amounts of functional.endogenous, rabbit immunoglobulins or functional endogenous, rabbit Igαand/or Igβ.

The present invention also provides other large transgenic animals,including but not limited to, birds, rodents and farm animals like cows,pigs, sheep, goats, donkeys, horses and the like expressing one or morehuman(ized) Ig loci and human(ized) Igα and/or Igβ. Again, preferably,these animals, additionally, do not produce substantial amounts offunctional. endogenous, immunoglobulins or functional endogenous, Igαand/or Igβ. Thus, these transgenic animals are capable of rearrangingthe human(ized) Ig loci and efficiently expressing a functionalrepertoire of human(ized) antibodies, with increased yields.

The invention is also directed to B-cells isolated from the differenttypes of transgenic animals described above, that express thehuman(ized) Igα and/or Igβ gene and the human(ized) immunoglobulin loci.Further, such B-cells can be immortalized using conventional methodsknown and used by skilled artisans in the field, including but notlimited to, using viral transformation, etc.

Immunization with antigen leads to the production of human(ized)antibodies against the same antigen in said transgenic animals.

Although preferred embodiments of the present invention are directed totransgenic animals having human(ized) Ig loci, it is to be understoodthat transgenic animals having primatized Ig loci and primatizedpolyclonal antisera are also within the spirit of the present invention.Similar to human(ized) polyclonal antisera compositions, primatizedpolyclonal antisera compositions are likely to have a reducedimmunogenicity in human individuals.

Once a transgenic non-human animal capable of producing diversifiedhuman(ized) immunoglobulin molecules is made (as further set forthbelow), human(ized) immunoglobulins and human(ized) antibodypreparations against an antigen can be readily obtained by immunizingthe animal with the antigen. A variety of antigens can be used toimmunize a transgenic host animal. Such antigens include, microorganism,e.g. viruses and unicellular organisms (such as bacteria and fungi),alive, attenuated or dead, fragments of the microorganisms, or antigenicmolecules isolated from the microorganisms.

Preferred bacterial antigens for use in immunizing an animal includepurified antigens from Staphylococcus aureus such as capsularpolysaccharides type 5 and 8, recombinant versions of virulence factorssuch as alpha-toxin, adhesin binding proteins, collagen bindingproteins, and fibronectin binding proteins. Preferred bacterial antigensalso include an attenuated version of S. aureus, Pseudomonas aeruginosa,enterococcus, enterobacter, and Klebsiella pneumoniae, or culturesupernatant from these bacteria cells. Other bacterial antigens whichcan be used in immunization include purified lipopolysaccharide (LPS),capsular antigens, capsular polysaccharides and/or recombinant versionsof the outer membrane proteins, fibronectin binding proteins, endotoxin,and exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,and Klebsiella pneumoniae.

Preferred antigens for the generation of antibodies against fungiinclude attenuated version of fungi or outer membrane proteins thereof,which fungi include, but are not limited to, Candida albicans, Candidaparapsilosis, Candida tropicalis, and Cryptococcus neoformans.

Preferred antigens for use in immunization in order to generateantibodies against viruses include the envelop proteins and attenuatedversions of viruses which include, but are not limited to respiratorysynctial virus (RSV) (particularly the F-Protein), Hepatitis C virus(HCV), Hepatitis B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

Therapeutic antibodies can be generated for the treatment of cancer byimmunizing transgenic animals with isolated tumor cells or tumor celllines; tumor-associated antigens which include, but are not limited to,Her-2-neu antigen (antibodies against which are useful for the treatmentof breast cancer); CD19, CD20, CD22 and CD53 antigens (antibodiesagainst which are useful for the treatment of B-cell lymphomas), (3)prostate specific membrane antigen (PMSA) (antibodies against which areuseful for the treatment of prostate cancer), and 17-1A molecule(antibodies against which are useful for the treatment of colon cancer).

The antigens can be administered to a transgenic host animal in anyconvenient manner, with or without an adjuvant, and can be administeredin accordance with a predetermined schedule.

After immunization, serum or milk from the immunized transgenic animalscan be fractionated for the purification of pharmaceutical gradepolyclonal antibodies specific for the antigen. In the case oftransgenic birds, antibodies can also be made by fractionating eggyolks. A concentrated, purified immunoglobulin fraction may be obtainedby chromatography (affinity, ionic exchange, gel filtration, etc.),selective precipitation with salts such as ammonium sulfate, organicsolvents such as ethanol, or polymers such as polyethyleneglycol.

The fractionated human(ized) antibodies may be dissolved or diluted innon-toxic, non-pyrogenic media suitable for intravenous administrationin humans, for instance, sterile buffered saline.

The antibody preparations used for administration are generallycharacterized by having immunoglobulin concentrations from 0.1 to 100mg/ml, more usually from 1 to 10 mg/ml. The antibody preparation maycontain immunoglobulins of various isotypes. Alternatively, the antibodypreparation may contain antibodies of only one isotype, or a number ofselected isotypes.

For making a human(ized) monoclonal antibody, spleen cells are isolatedfrom the immunized transgenic animal whose B-cells expressing theanimal's endogenous immunoglobulin have been depleted. Isolated spleencells are used either in cell fusion with transformed cell lines for theproduction of hybridomas, or cDNAs encoding antibodies are cloned bystandard molecular biology techniques and expressed in transfectedcells. The procedures for making monoclonal antibodies are wellestablished in the art. See, e.g., European Patent Application 0 583 980A1 (“Method For Generating Monoclonal Antibodies From Rabbits”), U.S.Pat. No. 4,977,081 (“Stable Rabbit-Mouse Hybridomas And SecretionProducts Thereof”), WO 97/16537 (“Stable Chicken B-cell Line And Methodof Use Thereof”), and EP 0 491 057 B1 (“Hybridoma Which Produces AvianSpecific Immunoglobulin G”), the disclosures of which are incorporatedherein by reference. In vitro production of monoclonal antibodies fromcloned cDNA molecules has been described by Andris-Widhopf et al.,“Methods for the generation of chicken monoclonal antibody fragments byphage display”, J Immunol Methods 242:159 (2000), and by Burton, D. R.,“Phage display”, Immunotechnology 1:87 (1995), the disclosures of whichare incorporated herein by reference.

In most instances the antibody preparation consists of unmodifiedimmunoglobulins, i.e., human(ized) antibodies prepared from the animalwithout additional modification, e.g., by chemicals or enzymes.Alternatively, the immunoglobulin fraction may be subject to treatmentsuch as enzymatic digestion (e.g. with pepsin, papain, plasmin,glycosidases, nucleases, etc.), heating, etc, and/or furtherfractionated to generate “antibody fragments”.

The present invention also includes pharmaceutical compositions orantibody preparations comprising the antibodies or their fragmentsobtained by the methods defined above The term “pharmaceuticallyacceptable ingredient” or “formulation” as used herein is intended toencompass a product comprising the claimed active ingredient(s), namelyhuman(ized) antibody or antibody fragment, in specified amounts, as wellas any product which results, directly or indirectly, from thecombination of the specified active ingredient(s) in the specifiedamounts. Such term is intended to encompass a product comprising theactive ingredient(s), and the inert ingredient(s) that make up thecarrier, as well as any product which results, directly or indirectly,from combination, complexation or aggregation of any two or more of theingredients, or from dissociation of one or more of the ingredients, orfrom other types of reactions or interactions of one or more of theingredients. Accordingly, the “pharmaceutical compositions” of thepresent invention encompass any composition made by admixing any activecompound of the present invention and a pharmaceutically acceptablecarrier.

The terms “administration of” and or “administering a” compound shouldbe understood to mean providing any active compound of the invention, inany formulation, to an individual in need of treatment.

The pharmaceutical compositions for the administration of the compoundsof this invention may conveniently be presented in dosage unit form andmay be prepared by methods well known in the art of pharmacy. Suitablemethods and carriers for use are those that are well-described in theart, and for example, in Remington, The Science and Practice ofPharmacy, ed. Gennaro et al., 20th Ed. (2000), although the skilledartisan in the field of immunology will readily recognize that othermethods are known and are suitable for preparing the compositions of thepresent invention. All methods include the step of bringing the activeingredient into association with the carrier which constitutes one ormore accessory ingredients. In general, the pharmaceutical compositionsare prepared by uniformly and intimately bringing the active ingredientinto association with a liquid carrier or a finely divided solid carrieror both, and then, if necessary, shaping the product into the desiredformulation. In the pharmaceutical composition the active ingredient isincluded in an effective amount, discussed above, sufficient to producethe desired effect upon the process or condition of diseases.Furthermore, formulations for the controlled, prolonged release ofantibody molecules have been described in U.S. Pat. No. 6,706,289, whosemethods are incorporated by reference herein.

Thus, the transgenic constructs, the vectors comprising the transgeneconstructs and the transgenic animals generated using the methodsdescribed above are all embodiments of the invention.

The invention is further illustrated, but by no means limited, by thefollowing examples.

EXAMPLE 1 Transfection of a Rabbit B-Cell Line with Human Igα and Igβ

To demonstrate the effect of human Igα and Igβ on the expression ofhuman mIgM in rabbit B-cells, such cells are transfected with expressionvectors encoding human Igα or Igβ or a human mIgM.

Human Igα and human Igβ and human IgM encoding genes are cloned inexpression vectors.

An immortalized rabbit B-cell line is transfected with the expressionvectors and cultured in medium in the presence of neomycin for theselection of stable transfectants. Resistant cells are analyzed by flowcytometry using antibodies specific for human IgM and human Igα and/orIgβ. Transfection of rabbit B-cells with an expression vector encodinghuman IgM results in low cell surface expression of human IgM.Cotransfection with human Igα and/or Igβ results in high cell surfaceexpression of human mIgM. This demonstrates that human Igα and/or β isnecessary and sufficient for high cell surface expression of human(ized)or chimeric mIgM.

EXAMPLE 2 Transfection of a B-Cell Line Derived From Any Animal WithHuman Igα and Igβ

To demonstrate the effect of human Igα and Igβ on the expression ofhuman mIgM in animal derived B-cells, expression vectors encoding humanIgα or Igβ or a human mIgM are transfected in B-cell derived fromchicken (DT40), cow, and pigs.

Immortalized B-cell lines are transfected with the expression vectorsand cultured in medium in the presence of neomycin for the selection ofstable transfectants. Resistant cells are analyzed by flow cytometryusing antibodies specific for human IgM and human Igα and/or Igβ.Transfection of rabbit B-cells with an expression vector encoding humanIgM results in low cell surface expression of human IgM. Cotransfectionwith human Igα and/or Igβ results in high cell surface expression ofhuman mIgM. This demonstrates that human Igα and/or Igβ, is necessaryand sufficient for high cell surface expression of human(ized) orchimeric mIgM.

EXAMPLE 3 Transgenic Rabbits Expressing the Humanized ImmunoglobulinLight and/or Heavy Chain Transgene With or Without Human Igα and/or Igβ

Transgenic rabbits were generated as described by Fan et al. (Pathol.Int. 49: 583-594, 1999). Briefly, female rabbits were superovulatedusing standard methods and mated with male rabbits. Pronuclear-stagezygotes were collected from oviduct and placed in an appropriate mediumsuch as Dulbecco's phosphate buffered saline supplemented with 20% fetalbovine serum. The exogenous DNA (e.g., expression vectors containinghuman(ized) immunoglobulin locus or human Igα or human Igβ) weremicroinjected into the pronucleus with the aid of a pair ofmanipulators. Morphological surviving zygotes were transferred to theoviducts of pseudopregnant rabbits. Pseudopregnancy was induced by theinjection of human chorionic gonadotrophin (hCG). Between about 0.1-1%of the injected zygotes developed into live transgenic rabbits.Integration of the transgene in the genome was confirmed by PCR andFISH.

The presence of antibodies containing human IgG and/or human kappa lightchain antigenic determinants in the serum of transgenic founder rabbitswas determined using an ELISA assay. Antibody expression on the surfaceof B-cells was analyzed by flow cytometry. Rabbits with a transgeneencoding a human(ized) immunoglobulin heavy chain locus, expressed 1-10ug/ml human IgM. Young animals (6-9 weeks) expressed 100-4000 ug/mlhuman IgG. However, the expression of human IgG declined rapidly tolevels of 10-100 ug/ml. Flow cytometric analysis of B-cells inperipheral blood revealed a small population of human mIgM+ cells(1-2%). The appendix of young rabbits contained up to 10% human mIgM+cells which disappeared rapidly with age.

Introduction of transgenes encoding human Igα and/or Igβ results in theexpression of 100-2000 ug/ml human(ized) IgM in serum and stableexpression of 2000-12000 ug/ml human(ized) IgG. In appendix 30-70% oflymphocytes are human(ized) mIgM+. In peripheral blood equivalentnumbers of B-cells express rabbit and human(ized) mIgM or mIgG.

All references cited throughout the disclosure along with referencescited therein are hereby expressly incorporated by reference.

While the invention is illustrated by reference to certain embodiments,it is not so limited. One skilled in the art will understand thatvarious modifications are readily available and can be performed withoutsubstantial change in the way the invention works. All suchmodifications are specifically intended to be within the scope of theinvention claimed herein.

What is claimed is:
 1. A method for isolating B-cells producing a humanIgα or a human Igβ subunit in a transgenic rabbit, comprising the stepsof: (a) microinjecting into pronuclear-stage zygotes, which werecollected from an oviduct of a superovulated female rabbit that had beenmated with a male rabbit, an expression vector for expression of anative full-length human Igα subunit of SEQ ID NO: 1 under control of aB-cell specific promoter/enhancer of CD79a or a native full-length humanIgβ subunit of SEQ ID NO: 7 under control of a B-cell specificpromoter/enhancer of CD79B; (b) transferring morphological survivingzygotes to the oviducts of pseudopregnant rabbits; (c) developing theinjected zygotes into live transgenic rabbits; (d) subjecting the livetransgenic rabbit to an antigenic stimulus; and (e) isolating B-cellsproducing said human Igα or human Igβ from the live transgenic rabbit ofstep (d).
 2. An isolated human antibody produced with the methodaccording to claim 1.